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Molecular and Cellular Biology, April 2000, p. 2890-2901, Vol. 20, No. 8
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

17beta -Estradiol Inhibits Apoptosis in MCF-7 Cells, Inducing bcl-2 Expression via Two Estrogen-Responsive Elements Present in the Coding Sequence

Bruno Perillo,1,* Annarita Sasso,1 Ciro Abbondanza,2 and Giuseppe Palumbo1

Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R., Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano", Facoltà di Medicina e Chirurgia, Università "Federico II", 80131 Naples,1 and Istituto di Patologia Generale ed Oncologia, Facoltà di Medicina e Chirurgia, Seconda Università di Napoli, 80138 Naples,2 Italy

Received 9 August 1999/Returned for modification 21 September 1999/Accepted 17 January 2000

We have found that 17beta -estradiol induces bcl-2 transcription in human breast cancer MCF-7 cells. To identify cis-acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected reporter constructs containing the bcl-2 major promoter (P1). Hormone inducibility was observed only when either of two sequences, located within the bcl-2 coding region and showing one and two mutations with respect to the consensus estrogen-responsive element, were inserted downstream from the P1 promoter. Both sequences behaved as enhancers exclusively in cells expressing the estrogen receptor and were able to bind this receptor in in vitro assays. Transfections into MCF-7 cells of plasmids carrying a bcl-2 cDNA fragment which included these two elements revealed that their simultaneous presence resulted in an additive effect on reporter gene activity, whose size resembled the increase of endogenous bcl-2 mRNA level observed in untransfected cells after hormone treatment. Moreover, the identified elements were able to mediate up-regulation of bcl-2 expression by 17beta -estradiol, since exogenous bcl-2 mRNA was induced by hormone challenge of MCF-7 cells transiently transfected with a vector containing the bcl-2 coding sequence cloned under the control of a non-estrogen-responsive promoter. Finally, we show that hormone prevention of apoptosis, induced by incubating MCF-7 cells with hydrogen peroxide, was strictly related to bcl-2 up-regulation. Our results indicate that the bcl-2 major promoter does not contain cis-acting elements directly involved in transcriptional control by 17beta -estradiol and that hormone treatment inhibits programmed cell death in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive elements located within its coding region.

* Corresponding author. Mailing address: Area della Ricerca---C.N.R., via P. Castellino, 111-80131 Naples, Italy. Phone: (39-081) 5797835. Fax: (39-081) 5607593. E-mail: perillo{at}unina.it.

Molecular and Cellular Biology, April 2000, p. 2890-2901, Vol. 20, No. 8
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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